The nitrogenase is comprised of two separately purified proteins, the Fe- protein and the MoFe-protein. Interactions between the Fe-protein and the MoFe-protein occur at many levels; during the maturation and assembly of nitrogenase and during the biological nitrogen fixation reaction. Both proteins acquire different conformations during these interactions. The specific aim of this research proposal is to elucidate the regions of the Fe-protein and the MoFe-protein that play important roles in the different conformations these proteins acquire for their interactions during the maturation and assembly of the nitrogenase and also during the biological nitrogen fixation reaction. Previously we have shown that the A. vinelandii UW97 is Nif due to a mutation, Ser44Phe, in the Fe- protein component. Utilizing this strain, we have isolated compensating suppressor mutations by using a genetic approach. The genetic approach was directed to isolate spontaneous Nif+ revertants. Characterization of two of these revertants showed that both of them have retained the original SerPhe mutation and carried second site compensating suppressor mutations in either the Fe-protein or in the and beta subunit of the MoFe-protein. During this grant period we proposed purify these mutated Fe-proteins and the mutated MoFe-protein and characterize their biochemical properties, and ability to interact with each other or with their wild type counterparts. We have also isolated targeted compensating mutations either in nifH, or nifK genes. This method involves propagating the cloned genes in the E. coli strain XL1-RED to introduce random mutations in these genes. By using this strategy, to date we have isolated several clones of A. vinelandii UW97 revertants. These clones were subjected to genetic analysis and confirmed that the compensating mutations of these suppressor mutants are located in the DNA fragments encoding nifH, nifD or nifK. Here we propose to identify exact locations of these compensating mutations by involving undergraduate students in this research, purify the altered nitrogenase from these strains, and characterize the functional properties by involving graduate students. The advantages of the approaches we have taken here is that they provide opportunity to unravel new regions of the Fe-protein and the MoFe-protein that are important for their interactions during maturation of the Fe-protein and biological nitrogen fixation reaction. We believe that these studies will provide new insights regarding how the components of this complex metalloenzyme maintain such high specificity in their interactions.